A limit of these techniques consists of the fact that it is difficult to obtain a high spatial resolution that would be useful, for example, for increasing the density of a biochip. This is because these techniques use a functionalized metallic layer, which must be illuminated with a collimated, and therefore not focused, light beam, otherwise there is a considerable loss of contrast (see the abovementioned article by S. A. Meyer et al.). It is not therefore possible to illuminate and observe a conventional plasmon sensor under optical microscopy. The other plasmon imaging techniques used with a parallel illuminating beam have, in all cases, a resolution limited to a few tens of microns; see, for example, Charles T. Campbell, Gibum Kim “SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics” Biomaterials 28 (2007) 2380-2392.
Document WO 2004/001399 and the article by D. Ausserré and M. P. Valignat “Surface enhanced ellipsometric contrast (SEEC) basic theory and λ/4 multilayered solutions”, Optics Express, Vol. 15, No. 13, Jun. 25, 2007, describe a high-contrast optical microscopy technique, known as “surface-enhanced ellipsometric contrast”, or SEEC, which is illustrated in FIG. 1. In accordance with this technique, a sample to be observed EO is deposited on a contrast-amplifying support SAC′, comprising a substrate SR, which is generally a reflecting substrate, and an antireflection layer or multilayer structure CA. The assembly made up of the sample and its support is illuminated by a spatially incoherent light beam F, linearly polarized by means of a polarizer P. The beam F may be annular; in any event, it is focused on the support-sample assembly by means of an objective LO, and its propagation axis is perpendicular to the surface of said support. In the case of an annular beam F, the light rays form, with the normal to the support, an angle of incidence of between θmin and θmax; if the beam is not annular, θmin→0°. The observation is made through a polarization analyzer A, generally oriented in such a way that its polarization axis is perpendicular to the direction of polarization of the beam F (it is then said that the polarizer P and the analyzer A are crossed); advantageously, the objective LO is also used for the observation. Thus, said objective and also the polarizer and the analyzer form a polarizing microscope.
The antireflection layer or multilayer structure CA is proportioned so as to minimize—ideally to cancel out—the light intensity which, reflected by the support SAC′, passes through the analyzer A. The object, which is generally transparent, disrupts the extinction condition, and therefore appears as a luminous form on a black background.
Strictly speaking, the extinction condition is satisfied for a single angle of incidence θ0, which would correspond to an annular illumination with θmin=θmax=θ0; however, obviously, under these conditions, the light intensity tends toward zero. More realistically, the interval θmaxθmin may be chosen to be about 5°. As a variant, the abovementioned documents indicate that it is possible to have recourse to a “conical” illumination (i.e. a nonannular illumination, characterized by θmin=0°), with θmax possibly reaching 20° or even 30°. In this case, the support SAC′ is proportioned on the basis of an average angle of incidence.
Under these conditions, the choice of the illumination conditions is a compromise, which cannot be entirely satisfactory. This is because:                an annular illumination is difficult to implement and involves a light intensity which is all the weaker the smaller the interval θmax−θmin;        a strongly converging conical illumination, with θmax of about 20° or more, results in a weak contrast since the extinction is actually obtained only at a single angle of incidence; and        a weakly converging conical illumination implies a small aperture of the objective, thereby limiting the spatial resolution with which the sample is observed.        
Like the SPR technique, the SEEC method can be applied to biological or biochemical analyses. For example, the support SAC′ can constitute the bottom of a Petri dish, in which case the observation must generally be made by immersion in a culture medium, which is sometimes difficult.
Document WO 02/50513 describes the application of the SEEC technique to the performing of ellipsometric measurements with spatial resolution, and also to the fabrication of biological sensors. In this case also, it is difficult to obtain both a high spatial resolution and a satisfactory contrast; moreover, the observation is sometimes difficult, in particular with regard to the applications to biology or to biochemistry.